Spatial distribution and insecticide resistance profile of Aedes aegypti and Aedes albopictus in Douala, the most important city of Cameroon

Prevention and control of Aedes-borne viral diseases such as dengue rely on vector control, including the use of insecticides and reduction of larval sources. However, this is threatened by the emergence of insecticide resistance. This study aimed to update the spatial distribution, the insecticide resistance profile of A. aegypti and A. albopictus and the potential resistant mechanisms implicated in the city of Douala. Immature stages of Aedes were collected in August 2020 in eight neighbourhoods in Douala and reared to adult stages. Adult bioassays, and piperonyl butoxide (PBO) synergist assays were carried out according to World Health Organization recommendations. Expression of some candidate metabolic genes including Cyp9M6F88/87, Cyp9J28a, Cyp9J10 and Cyp9J32 in A. aegypti, and Cyp6P12 in A. albopictus were assessed using qPCR. A. aegypti adults G0 were screened using real time melting curve qPCR analyses to genotype the F1534C, V1016I and V410L Aedes kdr mutations. Overall, A. aegypti is the predominant Aedes species, but analyses revealed that both A. albopictus and A. aegypti coexist in all the prospected neighbourhoods of Douala. High level of resistance was observed to three pyrethroids tested in both Aedes species. In A. aegypti a lower mortality rate was reported to permethrin (5.83%) and a higher mortality rate to deltamethrin (63.74%). Meanwhile, for A. albopictus, lower (6.72%) and higher (84.11%) mortality rates were reported to deltamethrin. Similar analysis with bendiocarb, revealed for A. aegypti a loss of susceptibility. However, in A. albopictus samples, analyses revealed a susceptibility in Logbessou, and confirmed resistance in Kotto (59.78%). A partial recovery of mortality was found to insecticides after pre-exposure to PBO. Cyp6P12 was found significantly overexpressed in A. albopictus permethrin resistant and Cyp9M6F88/87 for A. aegypti deltamethrin resistant. F1534C, V1016I and V410L mutations were detected in A. aegypti from different neighbourhoods and by considering the combination of these three kdr 14 genotypes were found. These findings provide relevant information which should be capitalised in the implementation of arbovirus vector control strategies and insecticide resistance management.

Introduction Aedes-borne viral diseases such as dengue, chikungunya, yellow fever and Zika are increasingly reported in Cameroon. Recently it was demonstrated that 13% of acute febrile patients consulting hospitals in Douala, Cameroon are due to dengue. During this study the co-circulation of three dengue serotypes, DENV-1, DENV-2, and DENV-3, were reported [1]. The viruses responsible for these diseases are transmitted by the infected bite of mosquitoes of the genus Aedes, whose major vectors are Aedes aegypti Linnaeus, 1762 and Aedes albopictus (Skuse), 1894. Aedes aegypti and A. albopictus are from different origins. A. aegypti originates from Africa [2] and is found throughout the tropics, while A. albopictus is native to Asia [3], but has invaded all the continents including Africa [4]. Both Aedes species are present in Cameroon with discrepancy in distribution. A. aegypti is distributed across the country while A. albopictus is restricted to the southern part of the country in latitude 6˚N where it tends to supplant the native species A. aegypti [5][6][7]. In cities where both species are found, A. aegypti is found most prevalent in downtown neighbourhoods with high building density while A. albopictus is mostly found in peri-urban neighbourhoods surrounded by vegetation [5,[8][9][10].
In absence of effective vaccine and specific treatment against this arboviruses, dengue fever prevention relies on vector control through reduction of larval sources and environment management, but during an epidemic, space spraying of insecticides is generally used to target adult mosquitoes [11]. Unfortunately, vector control is counteracted by the emergence and the continued development of resistance to all classes of insecticides used in public health [12].
Two main mechanisms are implicated in Aedes mosquitoes' resistance to insecticides: the metabolic resistance and the target site resistance. Metabolic resistance is mediated by upregulation of detoxification enzymes such as the monooxygenases (cytochrome P450s), glutathione S-transferases (GSTs) and carboxylesterases (COEs) [13]. Many genes of the P450 family, especially from the CYP9 and CYP6 subfamilies (CYP9J28, CYP9J10, CYP9J26, CYP6BB2 and CYP6P12) have been associated with resistance to pyrethroids [12,[14][15][16][17][18]. Target site resistance is caused by mutations in target genes such as the acetylcholinesterase (Ace-1), the GABA receptor and the voltage-gated sodium channel (VGSC) which causes knockdown resistance (kdr). The kdr is one of the main target site resistance mechanisms known as involved in the resistance for both pyrethroids and dichlorodiphenyltrichloroethane (DDT) insecticides [19][20][21]. In A. aegypti, 11 kdr mutations at 9 different codons positions in the VGSC domains I-IV have been reported [12,22]. Among them F1534C, V410L and V1016G, have already been identified in Cameroon [23][24][25]. Till date, only four VGSC mutations have been detected in A. albopictus affecting two codons (1532 and 1534). This study presents the spatial distribution and the insecticide susceptibility profile of A. aegypti and A. albopictus and the potential mechanisms implicated in the resistance of these arbovirus vectors in the city of Douala.

Insecticide resistance bioassays
Bioassays were performed according to WHO protocol using 2-5 days old G1 generation. Four replicates of 20-25 females per tube were exposed to 0.03% deltamethrin, 0.05% alphacypermethrin, 0.1% bendiocarb and 0.75% permethrin for 1hour. Mortality was recorded 24 hours later and mosquitoes alive or dead after exposure were stored in RNA later or silica gel, respectively. The resistance status was defined as follows: susceptible (mortality rate between 98-100%), probable resistance (mortality rate between 90-97%), and resistant (mortality rate inferior to 90%) [27].

Adult synergist assay with PBO
To evaluate the potential role of oxidase specific metabolic resistance mechanisms, synergist assays with 4% piperonyl butoxide (PBO) were performed. 2-5-days-old adults were preexposed for one hour to PBO-impregnated papers and after that immediately exposed to the selected insecticide. Mortality was scored 24 hrs later and compared to the results obtained with each insecticide without synergist according to the WHO standards [27]. The comparison of mortality rates after pre-exposure of mosquitoes to synergist and without pre-exposure to synergist was done using Chi-square test. The difference was statistically different when Pvalue was inferior to 0.05.

Knockdown resistance (kdr) genotyping
Three kdr mutations (V1016I, V410L and F1534C) were genotyped using around 30 samples per neighbourhood. Genomic DNA extracted from 30 individual mosquitoes per populations [28] were used for this experiment. Genotyping of the V1016I, V410L and F1534C mutations was performed by the real-time quantitative PCR using protocol published by Saavedra-Rodriguez et al. [29,30]. Each PCR reaction was performed in a 21.5 μl mixture containing 2 μl of DNA sample, 10 μl of SYBR1 Green (SuperMix), 1.25 μl of each primer and 5.75 μL of ddH2O. The thermocycle parameters were: 95˚C for 3 min, followed by 40 cycles of 95˚C for 20 s, 60˚C for 1min and 72˚C for 30 s and then a final step of 72˚C for 5 mins, 95˚C for 1 min, 55˚C for 30 s and 95˚C for 30 s.

Expression of detoxification Genes
RNA extraction and cDNA synthesis. For this experiment three groups of mosquitoes were used: the unexposed (control), the exposed (resistant) and the susceptible (laboratory susceptible strains). For each group three replicates of 10 mosquitoes per species were set up. RNA was extracted using the PicoPure RNA Isolation Kit (Arcturus1 Picopure RNA Extraction Kit Life Technologies, California, USA), following the manufacturer's recommendations. Quality and quantity of RNA obtained were assessed using a "NanoDrop Lite" spectrophotometer (Thermo Scientific Inc., Wilmington, USA) and was stored at -80˚C until further use.
Extracted RNA was used to synthesise complementary DNA (cDNA) using the Superscript III kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and the resulting cDNA was purified using a QIAquick spin column (QIAuick PCR Purification Kit, Qiagen) and diluted 2-fold to accommodate the volumes of the reaction.
Quantitative-reverse transcriptase PCR. Expression profiles of genes previously associated with A. aegypti or A. albopictus pyrethroid resistance [12,31] were assessed using quantitative reverse transcription PCR (qRT-PCR), in relation to two susceptible strains, Benin strain for A. aegypti [32,33] and Vector Control Research Unit (VCRU) strain for A. albopictus [34,35]. Standard curve analyses were performed for each primer pair to check the specificity and efficiency of amplifications. Four cytochrome P450 candidate genes were chosen for analysis in A. aegypti (Cyp9M6F88/87, Cyp9J28a, Cyp9J10 and Cyp9J32) and only one in A. albopictus (Cyp6P12). The reactions were performed in a mixture of 20 μL with 10 μL sybrGreen (Applied Biosystems, Texas, USA), 0.6 μL of each primer (10 μM), 7.8 μL of ddH2O and 1 μL of CDNA, under the following conditions: 95˚C for 3 min, followed by 40 cycles of 95˚C for 10 s and 60˚C for 10 s. The relative expression level and fold change (FC) of each candidate gene compared to susceptible strains were calculated using the 2-ΔΔCT method integrating the efficiency of PCR [36] after normalization with housekeeping genes: Aaeg60sL8, RPS3, RSP7 and qTubulin (S1 Table). All the primer sequences are presented in Table 1 respectively for A. albopictus and A. aegypti. The Mx Pro software integrated into the Agilent brand TaqMan machine was used.

Relative abundance and spatial distribution of A. aegypti and A. albopictus in Douala
A total of 13,927 specimens of Aedes spp. comprising 8,564 (61.49%) individuals belonging to A. aegypti species and 5,363 (38.51%) to A. albopictus species (Table 2) were collected during this study. Analysis revealed that both Aedes species coexist in all the prospected neighbourhoods in Douala but overall, A. aegypti is the predominant species (Fig 2). According to the environment (downtown vs suburban), A. aegypti is more predominant in all the neighbourhoods located in the downtown environment (Akwa, Brazzaville, Bépanda and Deïdo) while Table 1

Genes
Forward primer Reverse primer

Insecticide resistance profile of A. aegypti and A. albopictus in Douala
The results of adult bioassays showed a level of susceptibility varying according to the neighbourhood and the insecticide used. Three pyrethroids (0.03% deltamethrin, 0.75% permethrin and 0.05% alphacypermethrin) and one carbamate (0.1% bendiocarb) were used for adult bioassays.
Insecticide resistance profile of A. aegypti in Douala. All populations tested were resistant to three pyrethroids used with mortality rates varying from 23.30% (Brazzaville) to 63.74% (Yassa) to deltamethrin, from 6.57% (Brazzaville) to 55.45% (Yassa) to alphacypermethrin and from 5.83% (Brazzaville) to 60.16% (Yassa) to permethrin. With respect to bendiocarb, we observed resistance in Akwa and Yassa with mortality rates of 70.62% and 85.89% respectively. Probable resistance was observed in Brazzaville, Bonabéri and Logbessou with mortality rate between 91.79 and 94.91% (Fig 3).
Insecticide resistance profile of A. albopictus in Douala. All populations tested were resistant to all pyrethroids used with mortality rates varying from 6.72% (Deïdo) to 84.11% (Logbessou) to deltamethrin, from 24.35% (Kotto) to 71.21% (Logbessou) to alphacypermethrin, and from 59.26% (Deïdo) to 81.15% to permethrin. To bendiocarb, we observed susceptibility in Logbessou, probable resistance in Bonabéri and Yassa (mortality rate around 96%), confirmed resistance in Deïdo and Kotto (mortality rates of 74.17 and 59.78% respectively) (Fig 4). Tests with synergist PBO. To evaluate if the resistance observed in some populations was due by the Cyt-P450 genes, mosquitoes were pre-exposed to synergist PBO. The analyses revealed a significant recovery of susceptibility to insecticide with pre-exposure to PBO in most populations tested for both species. This was the case of A. aegypti from Logbessou where a recovery of susceptibility was reported to deltamethrin (84.11± 3.09% of mortality without PBO pre-exposure vs 97.28± 0.90% of mortality after PBO pre-exposure, P < 0.05), permethrin (81.15 ± 3.97% of mortality without PBO pre-exposure vs 99,10 ± 0,89% of mortality after PBO pre-exposure, P < 0.05), and alphacypermethrin (71.21 ± 8.69% of mortality without PBO pre-exposure vs 95,94 ± 1,73% of mortality after PBO pre-exposure, P < 0.05).
Expression of detoxification Genes. The qPCR analyses revealed that the only gene (Cyp6P12) whose expression was assessed in A. albopictus populations was found to be significantly overexpressed in the permethrin resistant Deïdo samples compared to susceptible VCRU lab strain (FC = 5.54 ± 0.73, P = 0.001); whereas in the deltamethrin-resistant Deïdo samples and the alphacypermethrin-resistant Yassa samples, the expression was not significantly different (Fig 5).
Two among the four Cyt-P450 genes assessed in A. aegypti were significantly overexpressed in field populations compared to susceptible Benin lab stain (Figs 6 and 7).  Knockdown resistance (kdr) genotyping. Three kdr mutations were genotyped F1534C, V1016I and V410L in eight locations of Douala (Table 3).
Concerning V1016I kdr genotyping, a total of 230 samples were examined. Among these, 131 (56.96%) were homozygote wild type (1016V/V), 76 (33.04%) were heterozygote (1016V/ I) and 23 (10%) were homozygote mutant (1016I/I). Overall, allelic frequency of homozygote wild type was 73.48% while for homozygote mutant was 26.52% (S1 Fig). For V410L kdr genotyping, we examined around 226 samples of which 125 (55.31%) were homozygote wild type (410 V/V), 78 (34.51%) were heterozygote (410V/I) and 23 (10.18%) were homozygote mutant (410I/I). Overall, allelic frequency of homozygote wild type was 72.57% while for homozygote mutant was 27.43% (S2 Fig). A total of 238 specimens were examined for F1534C genotyping. Overall allelic frequencies were 12.61% for homozygote wild type and 87.  (Fig 8). The frequency of each genotype varies according to the location. The only one susceptible genotype VV/VV/FF was present in five locations of the eight locations tested. The high frequency of this genotype was 37% (Yassa samples) followed by 7% (Kotto samples). The most predominant genotype VV/VV/CC was found in all the eight locations analyzed with frequency between 15 and 69% in Deïdo and Brazzaville respectively. Overall, the

Discussion
This study aimed to assess the spatial distribution, the insecticide resistance profile of A. aegypti and A. albopictus in Douala and to explore the potential resistance mechanisms involved.

Spatial distribution of A. aegypti and A. albopictus in Douala
Results showed that both species are present in all prospected neighbourhoods. Aedes aegypti was the predominant species in all neighbourhoods located in downtown while A. albopictus was more predominant in suburban localities. Previous studies reported the same observation in the city of Douala [5,6].
The predominance of A. aegypti in the downtown is consistent with previous works on the spatial distribution of A. aegypti and A. albopictus in other cities in Central Africa [8,10], in Asia [37] and in South America [38]. In these localities, it was clearly demonstrated that A. aegypti preferentially colonises man-made containers located in areas with high building density [6,37,39], whereas A. albopictus preferentially colonised breeding sites surrounded by vegetation [5,6,8]. In this study the predominant breeding sites were used tyres, which are described in   previous studies as preferential breeding sites for Aedes [8] because they offer not only very good shelter from predators but also from heat. The suburban neighbourhoods (Kotto, Logbessou, Yassa) which have a high vegetation cover compared to the downtown neighbourhoods (Deïdo, Akwa, Brazzaville and Bépanda) therefore offer a more favourable environment for the development of A. albopictus. This is consistent with previous studies conducted in some suburban neighbourhoods of Douala showing that A. albopictus is the predominant species in this environment [40]. Overall, analyses revealed that A. aegypti is the predominant species in the city of Douala, which corroborates previous observations [5,6]. This suggests that the environmental factors in Douala are more favourable to the development of this species in contrast to what is generally observed in localities in the southern part of Cameroon located below 6˚N [5].

Insecticide resistance profile of A. aegypti and A. albopictus
This study revealed that both species A. aegypti and A. albopictus are resistant to all insecticides tested (deltamethrin, alphacypermethrin, permethrin and bendiocarb). Indeed, resistance to permethrin 0.75% and deltamethrin 0.03% had already been reported in A. aegypti in Douala [23], in other cities in Cameroon [9,23], and other regions of the world such as  [9]. Globally, A. aegypti is more resistant to pyrethroids than A. albopictus. Similar results were obtained in other parts of the world including Malaysia [45]. However, these observations are different from those reported in the city of Yaoundé (Cameroon), indicating that the level of resistance observed in A. albopictus to pyrethroids was higher than in A. aegypti [9]. The resistance of A. aegypti and A. albopictus to pyrethroids could pose a serious threat to vector control programs, because pyrethroids are the main class of insecticide recommended for the control of adult Aedes mosquitoes notably in case of outbreaks [11].
A loss of susceptibility to bendiocarb was observed for both Aedes species. Resistance or loss of susceptibility observed to these insecticides (deltamethrin, permethrin and bendiocarb) in both Aedes species is difficult to explain because in Central Africa, specific insecticide treatments targeting Aedes spp. are currently very limited or non-existent [9,47]. This observation raises the question of the origin of the selection pressure by these species. As suggested previously [9,47], domestic use of insecticides through Indoor Residual Spraying (IRS) and impregnating bed nets, mass distribution of long-lasting insecticidal nets (LLINs) by national malaria control programme, and agriculture use could be the main source of resistance selection in Aedes vectors in Central Africa. Indeed, the use of pesticides in agriculture for the protection of market gardening could also promoted the emergence of resistance in mosquitoes by contamination of breeding sites and resting places of mosquitoes.

Resistance mechanism involved
Synergist assay with pre-exposure to PBO showed a partial or full recovery of susceptibility to all insecticides used in A. aegypti and A. albopictus. These observations are consistent with previous studies in Africa [9,18,23,43,48] and abroad [14,15]. This result indicates that the cytochrome P450 monooxygenases are playing the main role in the resistance of deltamethrin, alphacypermethrin, permethrin and bendiocarb. This result is supported by the overexpression of some genes analysed such as CYP6P12 in Deïdo in A. albopictus samples resistant to permethrin or Cyp9M6F88/87 in Logbessou and Brazzaville in A. aegypti samples resistant to deltamethrin and permethrin respectively. The CYP6 and CYP9 sub-families, were previously found to be associate with resistance to pyrethroids [14,15].
The F1534C mutation is common in A. aegypti and has a cosmopolitan distribution [12]. This mutation has been reported in West Africa including Ghana [21] and Burkina Faso [18,42]. Although not previously reported in Central Africa, previous work of Yougang et al. reported for the first time the existence of this mutation in A. aegypti in two cities in Cameroon [23]. The results of this work confirmed the presence of this kdr mutation in the city of Douala with a very high allelic frequency (87.39%). This high allelic frequency is similar to previous one reported in Douala [24]. The occurrence of these mutations is likely to impact the effectiveness of pyrethroid-based control measures against A. aegypti currently implemented in some African countries [51]. The combination of two of these mutations or all three mutations contribute strongly to increase resistance in A. aegypti. For example, V410L, alone confers low levels of resistance but co-evolved with V1016I or F1534C yielding higher levels of resistance [18,22,51]. Considering the tree kdr mutations together 14 genotypes were found, among them two were the most abundant: VV + VV + CC and VI + VL + CC (1016+410+1534). Similar observation was reported in Brazil [53]. Further studies are needed to establish whether there is an association between these mutations and phenotypic resistance in A. aegypti population from Cameroon.

Conclusion
This study revealed that A. aegypti and A. albopictus are present in city of Douala with A. aegypti as the most abundant. Both Aedes species are resistance to all insecticides tested and three kdr mutations have been detected in A. aegypti samples. A full or partial recovery of susceptibility observed after pre-exposure of mosquitoes to PBO, suggests a major role of P450 genes especially CYP6P12 and Cyp9M6F88/87 overexpressed in resistant mosquitoes. These findings are important for the control of Aedes mosquitoes in the city of Douala, but more studies need to be conducted to obtain additional information on other insecticide resistance mechanisms such as metabolic resistance.